DE69628912T2 - PRODUCTION AND USE OF A SPECIFIC GABA-AALPHA5 RECEPTOR LIGAND FOR TREATING THE DISEASE OF ALZHEIMER - Google Patents

Abstract

A method of treatment for enhancing cognition in the treatment of Alzheimer's disease, for example, employs a specific GABAA Alpha5 receptor ligand.

Description

The present invention provides the use of a compound for the manufacture of medicaments for Improvement of cognition (for example in the treatment of Dementia diseases such as Alzheimer's disease) and for such Suitable compositions.

A number of dementias How Alzheimer's is progressively worsening the cognition of those affected. It would be clear desirable, cognition in people in need of such treatment improve, for example for People suffering from dementia.

It was created by McNamara and Skelton in Psychobiology, 21: 101-108, reports that the inverse benzodiazepine receptor agonist βCCM improved spatial learning in the Morris water maze. However, β-CCM and other conventional inverse benzodiazepine receptor agonists anticonvulsant, which makes it clear that she not used as a cognitive enhancer in humans can be.

However, we have now found that it possible is to get medication that has cognitive enhancing effects have, but no anticonvulsant Have effects previously with inverse benzodiazepine receptor agonists have been described.

The benzodiazepine receptor site is located in the GABA _A receptor, a structure which is generally considered pentameric, with an integral chloride ion channel formed by the second transmembrane domain of each of the five subunits. Using modern molecular cloning techniques, a family of 14 GABA _A receptor subunits was identified in the mammalian brain, namely the α ₁ -, α ₂ -, α ₃ -, α ₄ -, α ₅ -, α ₆ -, β ₁ -, β ₂ , β ₃ , γ ₁ , γ ₂ , γ ₃ , δ and φ subunits. The selection of five subunits from a possible repertoire of 14 allows a variety of possible combinations, but the number of subtypes occurring and the extent of the GABA _A receptor heterogeneity remain unknown. If reference is made here in the following to an α ₁ -, α ₂ -, α ₃ -, α ₅ - or α ₆ -receptor, this naturally refers to the GAB _A α ₁ , GABA _A α ₂ , GABA _A α ₃ , GABA _A α ₅ and GABA _A α ₆ .

It has now been found that the use of an inverse α ₅ receptor agonist, which is relatively free of a certain activity at α ₁ and / or α ₂ and / or α ₃ receptor binding sites, can be used to to provide a drug that is suitable for improving cognition, but is not beneficial for convulsions.

Accordingly, the present invention provides the compound of a compound for the manufacture of a medicament for enhancing cognition in an individual without causing convulsions, the compound being an inverse α ₅ receptor agonist that is not a receptor agonist or inverse receptor Agonist at α ₁ and / or α ₂ and / or α ₃ receptors.

The individual becomes a mammal, on most suitable a person, and preferably a person who is under suffering from dementia such as Alzheimer's disease.

Thus, the compound used as an inverse agonist will bind to α ₅ receptors in concentrations at which there is no significant binding of an agonist or inverse agonist to α ₁ receptors. More appropriately, the compound will bind as an inverse agonist to α ₅ receptors in concentrations at which there is no significant binding of an agonist or inverse agonist to α ₁ and α ₂ receptors. Most suitably, the compound will bind as an inverse agonist to α ₅ receptors at concentrations at which there is no significant binding of an agonist or inverse agonist to α ₁ , α ₂ , and α ₃ receptors.

Binding of the inverse agonist to α ₅ can be partial (ie, the compound can be a partial agonist), but a fully inverse agonist is preferred at α ₅ . Likewise, the compound should not be a full or partial agonist or inverse agonist with the other receptors.

If desired, the compounds employed may have antagonist binding at α ₁ , α ₂ , α ₃ and α ₆ , after such binding will not interfere with the practice of the method of this invention. Regarding the relative binding to the different receptors, it is only the inverse agonist or agonist binding that is considered.

In general, such compounds will bind at least 10 times better to α ₅ receptors than to α ₁ receptors, ie the compound will be at least 10 times selective for α ₅ -containing receptors over α ₁ -containing receptors.

Suitably the compound for use in the invention is at least 10-fold selective for α ₅ -containing receptors over α ₁ - and α ₂ -containing receptors, more suitably at least 10-fold selective for α ₅ -containing receptors over α ₁ -, α ₂ - and α ₃ -containing receptors and most suitably at least 10 fold selective for α ₅ receptors over α ₁ -containing -, α ₂ -, α ₃ - and α ₆ -containing receptors.

Advantageously, the compound for use in the invention is at least 25-fold selective for α ₅ -containing receptors over α ₁ -containing receptors, e.g. B. 25-fold selective for α ₅ -containing receptors compared to α ₁ - and α ₂ -containing receptors, more suitable at least 25-fold selective for α ₅ -ent holding receptors to α ₁ -, α ₂ - and α ₃ -containing receptors and most suitably at least 25-fold selective for α ₅ -containing receptors to α ₁ -, α ₂ -, α ₃ - and α ₆ -containing receptors.

Preferably, the compound for use in the invention is at least 50-fold selective for α ₅ -containing receptors over α ₁ -containing receptors, e.g. B. α ₁ - and α ₂ -containing receptors, more suitable at least 50-fold selectively for α ₅ -containing receptors compared to α ₁ -, α ₂ - and α ₃ -containing receptors and most suitably at least 50-fold selective for α ₅ -containing receptors against α ₁ -, α ₂ -, α ₃ - and α ₆ -containing receptors.

Advantageously, the compound for use in the invention will be at least 100-fold selective for α ₅ -containing receptors over α ₁ -containing receptors.

The preferred use of this Invention has the additional Advantage of being able to improve cognition without unwanted anxiogenic To induce effects.

A preferred receptor for determining the α ₅ binding is the α ₅ β ₃ γ ₂ receptor. A preferred receptor for determining the α ₁ binding is the α ₁ β ₃ γ ₂ receptor. A preferred receptor for determining the α ₂ binding is the α ₂ β ₃ γ ₂ receptor. A preferred receptor for determining the α ₃ binding is the α ₃ β ₃ γ ₂ receptor. A preferred receptor for determining the α ₆ binding is the α ₆ β ₃ γ ₂ receptor.

Receptors are used in the international Patent Application No. WO 92/22652 and WO 94/13799.

The compounds for use in of this invention by screening against the receptors identified above using identified by methods known in the art. preferred Techniques include those described in Goeders et al. described (see here hereinafter).

Determining whether the connections Receptor agonists, partial receptor agonists or inverse receptor agonists are alike using methods known in the art. Preferred techniques include those described in Wafford et al. described (see here below).

Particularly suitable compounds for use in this invention will have a Ki (nM) against the α ₅ β ₃ γ ₂ receptor of less than 5, more suitably less than 2 and most suitably less than 1, for example about 0.5.

Particularly suitable compounds for use in this invention will have a Ki (nM) versus α ₁ β ₃ γ ₂ receptor of more than 10, more suitably more than 20, most suitably more than 40, for example about 50.

Particularly suitable compounds for use in this invention will have a Ki (nM) over α ₂ β ₃ γ ₂ receptor of more than 5, more suitably more than 10, most suitably more than 20, for example about 25.

Particularly suitable compounds for use in this invention will have a Ki (nM) over α ₃ β ₃ γ ₂ receptor of more than 5, more suitably more than 10, most suitably more than 20, for example about 25.

Particularly suitable compounds for use in this invention will have a Ki (nM) versus α ₆ β ₃ γ ₂ receptor of more than 10, more suitably more than 20, most suitably more than 40, e.g. about 80.

To develop their activity without the compounds intravenous have to be administered the compounds for use in this invention are the most preferably able to cross the blood-brain barrier after oral administration to happen.

A connection that the above executed desirable Possesses properties that the usefulness of owning these Features shows is FG 8094.

The present invention also provides a pharmaceutical composition for use in improving cognition without causing convulsions, which is a compound that is an inverse α ₅ receptor agonist that is not a receptor agonist or inverse receptor agonist for α ₁ - and / or α ₂ - and / or α ₃ receptors, and a pharmaceutically acceptable carrier therefor.

The compositions are the most suitable for the oral administration adapted to humans, although parenteral routes of administration can also be considered, for example by intravenous, intramuscular or subcutaneous administration or topical or rectal.

For oral use of the cognitive enhancer can selected Compound for example in the form of a tablet or pharmaceutical acceptable vehicle or diluent, optionally with known adjuvants, such as. B. alum, in one pharmaceutical composition according to standard pharmaceutical practice be administered. The compounds can be administered orally, parenterally, including by intravenous, intramuscular, administered intraperitoneally or subcutaneously, or topically become.

For oral use, the cognitive enhancer can be administered, for example, in the form of tablets or capsules or as an aqueous solution or suspension. In the case of tablets for oral use, carriers commonly used include lactose and corn starch and lubricants such as. As magnesium stearate, are usually added. For oral administration in capsule form include suitable nete diluent lactose and dried corn starch. If aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners and / or flavors can be added.

For the intramuscular, Intraperitoneal, subcutaneous and intravenous use usually become sterile solutions of the active ingredient and the pH of the solutions should be set and buffered in a suitable manner. For intravenous use the total concentration of the dissolved substances should be set, around the preparation to make isotonic.

For topical administration may be the cognitive enhancer for example as a suspension, lotion, cream or ointment using a pharmaceutically acceptable carrier such as e.g. B. water, mixtures of water and water-miscible solvents such as lower ones Alkanols, vegetable oils and polyalkylene glycols.

The pharmaceutical preparation can also be non-toxic auxiliary substances, e.g. B. emulsifying, preserving, Wetting agents, thickeners and the like, such as. B. the polyethylene glycols 200, 300, 400 and 600, the carbowaxes 1,000, 1,500, 4,000, 6,000 and 10,000, antibacterial components such as quaternary ammonium compounds, Phenylmercury salts, which are known to be cold sterilizing Have properties and which are not harmful when used, Thimerosal, methyl and propyl paraben, benzyl alcohol, phenylethanol, buffering components such as sodium chloride, sodium borate, sodium acetates, Gluconate buffer and other conventional Ingredients such as sorbitan monolaurate, triethanolamine, oleate, polyoxyethylene sorbitan monopalmitylate, Contain dioctyl sodium sulfosuccinate, monothioglycerin, thiosorbitol and ethylenediaminetetraacetic acid.

If a cognitive enhancer The daily dose is used in a human individual normally be determined by the prescribing doctor, whereby the dose generally depends on the age, weight and reaction of the individual patient as well as the severity of the patient's symptoms will vary. However, in most cases it will be an effective daily dose in the range of about 0.005 mg / kg to about 100 mg / kg body weight and preferably from 0.05 mg / kg to about 50 mg / kg, e.g. B. of about 0.5 mg / kg to about 20 mg / kg body weight, administered in single or divided doses. In some make however, a dose may be outside of these limits.

Generally dosage unit forms are used for the oral administration 1 to 800 mg, more usually 2.5 to 250 mg, preferably 5 to 100 mg, for example 10, 20 or 50 mg.

A preferred cognitive enhancer Agent for use in this invention is 9H-imidazo [1,5-a] pyrrolo [2,1-C] [1,4] benzodiazepine-1-carboxylic acid-11,12,13,13a-tetrahydro-7-methoxy- 9-oxo-ethyl ester, (S) - (9CI). The making of this connection is done in the Canadian Application CA 1266671 A2 900313 (divisional application of the Canadian patent application No. 503 329). This connection is sometimes called FG 8094 known.

The compositions according to the invention can be used in any suitable manner, for example by conventional ones Tableting or capsule filling techniques, getting produced.

This invention also provides a method of identifying a compound that is capable of cognitive enhancement without causing convulsions, the method using α ₅ - and α ₁ - and / or α ₂ - and / or α ₃ - and / or α ₆ receptors to determine whether the compound is a ligand at α ₅ -, but not at α ₁ - and / or α ₂ - and / or α ₃ - and / or α ₆ receptors, and around determine whether the binding to α ₅ receptors is that of an inverse agonist and to determine whether binding to the α ₁ and / or α ₂ and / or α ₃ and / or α ₆ receptors is binding Is agonists or inverse agonists.

This invention provides a method of finding a compound capable of improving cognition without causing cramps, which method uses a GABA _A -binding receptor to determine that the compound is an inverse α ₅ agonist and none Agonist or inverse agonist at α ₁ and / or α ₂ and / or α ₃ .

This invention also provides one Ligands ready, which was previously unknown in is able to improve cognition without having cramps caused by a detection method of this invention was identified.

This invention also provides pharmaceutical composition ready which one according to the method compound identified in this invention.

The following references provide useful background information:
Goeders, NE, and Kuhar, MJ (1985), Benzodiazepine binding in vivo with [ ³ H] Ro, 15-1788, Life Sci. 37: 345-355.

McKernan, RM, Quirk, K., Prince, R., Cox, PA, Gillard, NP, Ragan, CI, and Whiting, PJ (1991), GABA _A receptors immunopurified from rat brain with α-subunit specific antibodies have unique pharmacological properties, Neurons 7: 667-676.

Quirk, K., Gillard, N.P., Ragan, C.I., Whiting, P.J., and McKernan, R.M. (1994), Y-Aminobutyric acid Type A receptors in the rat brain can contain both Y2 and Y3 subunits but Y1 does not exist in combination with another Y-subunit, Mol. Pharmacol. 45: 1061-1070.

Wafford, KA, Whiting, PJ, and Kemp, JA (1993), Differences in affinity and efficacy of benzodiazepine receptor ligands on recombinant GABA _A receptor subtypes, Mol. Pharmacol. 43: 240-244.

Whiting, PW, Wafford, K., and McKernan, RM (1996), GABA _A receptors in the central nervous system, International Reviews in Neurobiology.

Wisden, W., Herb, A., Wieland, H., Keinanen, K., Luddens, H., and Seeberg, P.H. (1991), Febs Lett. 289, 227-230.

EXAMPLE 1

FG 8094 was suspended in 0.5% carboxymethyl cellulose solution, to provide an injectable solution.

EXAMPLE 2

The following examples illustrate pharmaceutical compositions according to the invention.

Tablets containing 1-25 mg FG 8094

Tablets containing 26-100 mq FG 8094

FG 8094, cellulose, lactose and a Part of the cornstarch are mixed and granulated with 10% corn starch paste. The resulting Granules are sieved, dried and mixed with the rest of the cornstarch and Magnesium stearate mixed. The resulting granules are then pressed into tablets, the 1.0 mg, 2.0 mg, 25.0 mg, 26.0 mg, 50.0 mg and 100 mg of the active Connection included per tablet.

EXAMPLE 3

The exemplary FG 8094 (1.5 mg) was combined with hydroxypropylmethyl cellulose (58.5 mg) to give a to form a small pellet. The pellets were implanted subcutaneously in rats. The resulting FG 8094 blood levels were for more than 6 hours higher than 100 ng / ml.

General procedures

1. Radioligand binding studies

The radioligand binding studies were performed using membranes prepared from cells stably transfected with the following subunit combinations: α ₁ β ₃ γ ₂ , α ₂ β ₃ γ ₂ , α ₃ β ₃ γ ₂ , α ₅ β ₃ γ ₂ , α ₆ β ₃ γ ₂ . Incubations were performed using 20-100 mg membrane protein in a total volume of 0.5 ml in 10mM Tris-HCl, 1mM EDTA, pH 7.4 for one hour at room temperature before termination through Whatman GF / C filters followed by 3 x 3 ml washes with 5 mM Tris-HCl, pH 7.5, and scintillation counting. [ ³ H] Ro 15-1788 was used as the radioligand to GA To label BA _A receptors, with the exception of cells expressing α ₆ β ₃ γ ₂ , in which [ ³ H] Ro 15-4513 was used, since this type of receptor [ ³ H] Ro 15-1788 was not of high affinity binds. The non-specific binding was determined with 10 μM Ro 15-4513 and the radioligands were used in concentrations which corresponded to twice their Kd values for each subtype. The Ki values were calculated using the Cheng-Prussof equation.

2. In vivo [ ³ H] Ro 15-1788 binding in the mouse brain

The in vivo binding of [ ³ H] Ro 15-1788 to mouse brain was essentially as described by Goeders and Kuhar (1985). Male Swiss Webster mice received a 0.9% NaCl solution containing 50 mCi / kg [ ³ H] Ro 15-1788 (0.05 ml / 10 g body weight) via the tail vein. The animals were sacrificed by decapitation three minutes later, at which time peak levels of [ ³ H] Ro 15-1788 were measurable in the brain (data not shown). 30 minutes before sacrifice, FG 8094 (0.3, 1.0, 3.0 and 10.0 m / kg) or diazepam (30.0 mg / kg to define non-specific binding) was intraperitoneally in 0.5 % Carboxymethylcellulose (CMC) given. Each mouse brain was then quickly removed, homogenized in 5.0 ml 50 mM Tris-HCl, 5.0 mM EDTA, pH 7.4 at 4 ° C using an Ultra-Turrax homogenizer for 10 s at setting 5. In a separate series of experiments, different areas of the brain, selected for their preferred expression of certain GABA subtypes, were also examined. Binding of [ ³ H] Ro 15-1788 to the cerebellum is almost exclusively α ₁ -like (Quirk et al., 1994), while binding to the spinal cord indicates binding to receptors which are α ₂ - or α ₃ - Contain subtypes, α ₁ , α ₄ and α ₅ are rare or absent in this region (Wisden et al., 1992). The brains were quickly separated into the forebrain and cerebellum and the spinal cord was also removed. Each forebrain was homogenized in 4.0 ml buffer (approximately 14 ml / g wet weight of tissue), each cerebellum was homogenized in 1.0 ml buffer (approximately 14 ml / g wet weight of tissue) and each spinal cord in 1.0 ml buffer (about 20.0 mg / g wet weight of tissue). Homogenate volumes (3 x 200 ml) were quickly filtered through GF / B filters, washed with 2 x 5.0 ml volumes of ice-cold buffer and the remaining radioactivity counted by liquid scintillation spectroscopy.

3. Electrophysiological Studies - functional Effects of FG 8094

Binding studies with transfected cells containing α ₁ β ₃ γ ₂ S, α ₂ β ₃ γ ₂ S and α ₅ β ₃ γ ₂ S have identified compounds with α ₅ selectivity. These compounds were studied using electrophysiological techniques (Wafford et al., 1993) to determine their functional effects on α ₁ β ₂ γ ₂ S-, α ₂ β ₂ γ ₂ S-, α ₃ β ₂ γ ₂ S-, α Determine ₅ β ₂ γ ₂ S and α ₆ β ₂ γ ₂ S-GABA _A receptors that had been expressed in Xenopus oocytes. Oocyte nuclei were injected directly with 10.0–20.0 nl of the relevant cDNAs (6.0 ng / μl) that had been genetically engineered into the expression vector pCDM8. After incubation for 24 h, the oocytes were placed in a 50 ml bath and rinsed with saline. Using the two-electrode voltage clamp recording method, the GABA-activated currents could be measured by bathing the cell in a GABA-containing solution.

First, a reproducible current response was established using a GABA concentration that gave about 20% of the maximum GABA response that could be caused (EC ₂₀ ). Co-administration of the test compound with this EC ₂₀ concentration to GABA led to a modulation of the reaction via an action at the BZ binding site. The reaction could be potentiated by an FC receptor agonist or inhibited by an inverse BG receptor agonist.

4. Seizures and cognitive studies

drug agents

FG 8094 was developed by the group of medicinal chemistry from Merck, Sharp and Dohme Neuroscience Research Center's synthesized and suspended in 0.5% CMC. Vehicle or FG 8094 was i.p. 30 Administered minutes before the start of the first training attempt.

Spasm promotion test

(i) acute treatment

Sixty male Sprague Dawley rats (225-265 g), housed in groups of four with freely accessible feed and water, served as subjects. The rats were assigned to one of six groups and received: vehicle or 0.5, 1.0, 2.0, 4.0 mg / kg FG 8904 or 10.0 mg / kg des non-selective inverse receptor agonist CGS 8216, a known anticonvulsant. Vehicle, FG 8094 or CGS 8216 was administered ip 30 minutes before an intrusion of the anticonvulsant pentylene tetrazole (PTZ, 40 mg / ml). PTZ was injected into the rat tail vein at a rate of 1.0 ml / min. infused. The tendency for clonic and tonic seizures was recorded and the respective threshold was calculated using the equation: threshold dose = infusion rate (ml / s) × PTZ (mg / ml) × (1000 / rat weight) × latency (s).

(ii) chronic treatment

The used in this study Rats received chronic treatment with FG 8094 during one MWM experiment. The rats received an intraperitoneal dose once a day (Monday to Friday) and received a total of 24 cans. After each drug administration, the rats were treated in one MWM tested and received the last dose of drug 48 Hours before the cramp stimulation test. The subjects were one of four treatment groups (n = 8) and received either vehicle, 0.1, 0.3 or 1.0 mg / kg FG 8094.

On the day of the cramp stimulation test the rats were held and pentylene tetrazole (PTZ, 40.0 mg / ml) was infused as described above. The latency (s) up to both clonic and tonic (full scale) cramps recorded. The latency data was transformed to the The dose of PTZ required to reach the threshold of both clonic and tonic seizures to reach.

Cognitive studies

animals

male PVG hood rats (Bantin & Kingman, Hull, V.K.) weighing about 300 g were in groups of four housed with free access to food and water. The Animals were kept on a 12/12 h light-dark cycle, the lighting was switched on at 07:00 h.

apparatus

The Morris Water Maze is there from a white, circular Fiberglass pool with a diameter of 2 m, filled with a cloudy mixture of water and white Dyestuff (E308, Morton International), kept at 26-28 ° C becomes. The pool was in the middle of a quiet room housed on its walls Images with high-contrast black and white patterns are shown as spatial "extra labyrinth" references were. "North" was roughly determined and the pool divided into four equal quadrants "Northeast", "Southeast", "Southwest" and "Northwest". A hidden platform (13 × 13 cm), the 2 cm below the water surface was immersed, was placed in the center of the NE quadrant.

A video camera with closed Circuit, which was equipped with a wide-angle lens directly above mounted in the middle of the pool and connected to an image analyzer, who digitized the image. The digital information became one Archimedes microcomputer headed, ran on the "water maze", a software package, purchased from Paul Fray LTD (Cambridge, V.K.). The software package provided the following measurements: Latency until reaching the platform, length of the path taken, average swimming speed, time and total distance spent in the target quadrant was covered.

training

The rats were assigned to four treatment groups (n = 8): vehicle, 0.1, 0.3, 1.0 mg / kg FG 8094. During the learning phase of the experiment, four experiments per day were carried out with all groups. During this time, the hidden platform was submerged in the NE quadrant of the water maze. The rat was taken out of the home cage and brought into the water labyrinth at one of four quasi-arbitrary locations ("North", "East", "South" or "West"), her head pointing to the pool wall and almost touching it. The trials started when the rat was released by the experimenter and ended when the rat climbed onto the platform and the mean escape latency was recorded. The maximum test duration was 60 s. If the rat had not climbed onto the platform after this time, the experiment ended automatically, the experimenter placed the rat on the platform and an escape latency of 60 s was recorded. The rat remained on the platform for 30 s and was then placed in an opaque plastic container with high side walls for another 30 s (interval between attempts, ITI). At the end of the ITI, the rat was brought back into the pool, but on one their location, and after being released the next attempt began. This procedure was repeated until four attempts were completed.

Learning and memory tests

exploratory trials

The flight latency usually decreases in the course of several training days from 60 s to about 20 s. A "probing attempt" assesses the spatial memory the rat for the location of the hidden platform. During this attempt removed the platform from the water maze and allowed the rat to crawl the pool for 60 s before taking it out of the pool has been. In case of treatment rat memory for location platform, it will spend a long time search the quadrant where the platform was previously hidden was (NE) as a rat receiving vehicle treatment.

Based memory

Performance deficits that are drug-induced Side effects (such as blurred vision or motor deficits, that impaired one floating assets to lead) could have been based by means of Memory detected. In this procedure, the platform also placed in the NE quadrant, but 3 cm above the Water level raised and thus visible to the rat. if the Treatment does not affect mean escape latencies up to the visible platform has any group differences in the middle flight latency to an invisible platform one Impact on the cognitive process can be attributed. The training scheme was identical to the training scheme used for the learning training was, and was continued until the mean escape latencies did not differ significantly between the groups.

memory

Six days after the probing attempt, while what time the rats were not exposed to the water maze or FG 8094, the rats were reapplied the learning protocol with the one immersed in the NE quadrant Platform tested.

reversal learning

The reverse training was with the learning phase of the experiment identical, except that the platform is in the middle of the SW quadrant was submerged.

Results

1. Selectivity of FG 8094 for α ₅ β ₃ γ ₂

Competitive curves for each of the cell lines show that FG 8094 is selective for receptors that contain the α ₅ subunit (see Table 1). FG 8094 was 107-fold selective for α ₅ -containing receptors against α ₁ , 60-fold selective against α ₂ , 53-fold selective against α ₃ and 184-fold selective against α ₆ . This represents a new pharmacological profile, since most selective FCs, such as. B. Zolpidem or CL 218872, have a greater affinity for α ₁ - than for α ₅ -containing receptors (Pritchett and Seeberg, 1990). FG 8094 has a greater preference for α ₅ -containing receptors than α ₁ -containing receptors than any other compound previously reported.

2. Occupation of GABA _A subtypes based on the displacement of [ ³ H] Ro 15-1788 binding from the whole brain by FG 8094 in vivo

Three minutes after [ ³ H] Ro 15-1788 was administered intravenously to mice, there were significant levels of radioactivity in the brain. It was estimated that more than 95% of [ ³ H] Ro 15-1788 was associated with membranes because more than 95% of the radioactivity was retained after filtration of the homogenates (data not shown).

Diazepam (30.0 mg / kg) displaced 90% of the [ ³ H] Ro 15-1788 binding in the brain, indicating that ligand present in the membranes was bound to GABA _A receptors. When administered intraperitoneally in doses of up to 3.0 mg / kg, FG 8094 did not significantly displace the specific [ ³ H] Ro 15–1788 binding (Da not shown). This implies that the number of any other GABA _A receptor subtype populated at these doses should be small and below the detection limit in this assay. Since FG 8094 has the highest affinity for α ₅ -containing receptors (Table 1) and these represent less than 5% of the total GABA _A receptor population, it is possible that doses ≤ 3.0 mg / kg FG 8094 occupy this subtype. At 10.0 mg / kg, FG 8094 significantly displaced [ ³ H] Ro 15–1788 binding (p <0.05). Receptors that contain α ₁ -, α ₂ - and α ₃ subunits form the three largest subtypes in the brain and make up about 90% of the GABA _A receptors (McKernan et al., 1991, Whiting et al., 1994) , At this dose, FG 8094 is likely to occupy predominantly α ₂ - and α ₃ -containing receptors (for which it has a higher affinity than for α ₁ -containing receptors). The receptors which are not displaced by FG 8094 at 10.0 mg / kg are most likely to contain receptors containing an α ₁ subunit which comprise 40-50% of all GABA _A receptors in the brain and the lowest affinity for FG 8094 Has.

The occupation of α ₂ -, α ₃ - and α ₁ -containing receptors 30 minutes after the administration of FG 8094 (1.0 mg / kg) was examined more directly by determining the displacement of the [ ³ H] Ro 15-1788 binding in a brain region that is enriched for these subtypes. The cerebellum was used as an α ₁ -selective tissue and the spinal cord as an α ₂ / α ₃ -selective tissue. The forebrain, which contains most of the subtypes of GABA _A receptors, was also included. The inhibition of in vivo binding of [ ³ H] Ro 15-1788 30 minutes after administration of FG 8094 (1.0 mg / kg) is shown in Table 2.

Assuming that the percent inhibition of [ ³ H] Ro 15-1788 binding in vivo is a measure of the receptor occupation, the concentration at the receptor site in the brain can be calculated according to Clarke's equation (occupation = [drug ingredient] / [drug ingredient] + Ki). Substituting the at α ₁ - and α _{2/3 of} -containing receptors certain cast, the concentration of FG 8094, which is available at BZ binding sites in rat brain, 7.8 nM and 13.2 nM. The population of α ₅ -containing receptors cannot be measured directly due to the low frequency of the receptor, but can be calculated using Clarke's equation using the drug concentration and binding affinity.

This predicts a 95-97% occupancy of α ₅ -containing receptors 30 minutes after administration of FG 8094 (1 mg / kg).

3. Electrophysiological Studies - Functional Effects of FG 8094

The modulatory effect on the GABA-EC ₂₀ value is in 1 shown for FG 8094 at 1 μM, which would be almost maximum for all receptor subtypes examined, and for two standard compounds, flunitrazepam (1 μM, a full receptor agonist) and DMCM (1 μM, a full inverse receptor agonist), at concentrations which would be maximal in binding studies with α ₁ β ₂ γ ₂ S, which shows that FG 8094 is a full inverse receptor agonist with α ₅ β ₂ γ ₂ . 2 compares the effects of FG 8094 on receptors containing another α subunit and demonstrates that it has a much lower potency on other α subunit combinations. It has no intrinsic effect on α ₁ β ₂ γ ₂ , suggesting that it is a low affinity antagonist, and it is a partial inverse receptor agonist on α ₂ β ₂ γ ₂ - and α ₃ β ₂ γ ₂ - receptors. A comparison of the efficacy when using a standard concentration of the drug at α ₁ β ₂ γ ₂ S demonstrated that FG 8094 is functionally selective for α ₅ -containing receptors and that its effects are mainly exerted via this subtype.

4. Seizures and cognitive studies

(I) Acute Treatment - A one-way analysis of variance the mean threshold doses for tonic and clonic seizures a major effect on tonic [F (5.49) = 3.70; p = 0.007], but not clonic [F (5.48) = 2.01; p = 0.09)] seizures. Post hoc tests reveal that only CGS 8216 significantly reduced threshold doses for tonic seizures, which shows that it an antispasmodic Had effect.

(I) Chronic Treatment - An Analysis of Variance revealed no significant treatment effect on the threshold dose from PTZ for either clonic [F (3.25) = 0.23; p = 0.87] or tonic [F (3.25) = 0.06; p = 0.98] seizures. These results demonstrate that that used in the study Chronic treatment protocol does not increase sensitivity to the anticonvulsant Funds PTZ led.

learning trials

3a illustrates the mean escape latencies during the nine-day training period. An analysis of variance of the mean swimming speed during the flight to the hidden platform revealed no significant dose effect [F (3.28) = 0.87], no significant effect of the day [F (8.216) = 1.44, p> 0.1] and no dose-day interaction [F (24.216) = 1.04, p> 0.4]. A variance analysis of the mitt Earlier escape latencies with treatment and day factors revealed a major treatment effect [F (3.28) = 3.01, p <0.05], a significant day effect [F (8.216) = 34.67, p < 0.001], but no dose-day interaction [F (24.216) = 0.75, p> 0.5]. Although the mean escape latencies of groups receiving 0.3 and 1.0 mg / kg FG 8094 appear to be shorter than that of the vehicle control group or the 0.1 mg / kg group, the Newman observation Keul tests not confirmed post hoc.

exploratory trials

Analysis of the variance of the percentage of time spent swimming in the northeast quadrant revealed no significant dose effect [F (3.28) = 0.18] (see 3b in terms of details), which implies that memory or learning efficiency did not differ among the groups.

Supported memory

A variance analysis of the mean escape latency to reach the visible platform showed no significant dose effect [F (3.28) = 0.54]. However, there was a significant dose effect on the mean path length [F (3.28) = 4.27, p <0.05]. Post-hoc Newman-Keuls tests showed that the vehicle group traveled significantly longer than all groups given FG 8094 ( 3c ).

memory test

A variance analysis of the mean escape latency revealed a significant dose effect [F (3.28) = 3.53, p <0.05]. A Newman-Keuls test post hoc showed that the mean escape latency of the vehicle group was longer than that of any of the FG 8094 groups (p <0.05). These data suggest that the groups receiving FG 8094 retained spatial information regarding the location of the hidden platform better than the vehicle control group (see 3d ).

reversal learning

While Reverse Learning became the platform in the middle of the Southwest Quadrant laid. A variance analysis of the mean escape latencies revealed a significant dose effect [F (3.28) = 3.31, p <0.05], a significant Experimental effect [F (3.84) = 9.53, p <0.001], but not significant Interaction [F (9.84) = 1.66, p> 0.1]. A Newman-Keuls test post hoc indicated that the mean escape latency of the vehicle group significantly longer than that of all FG 8094 groups was (p <0.05). On subsequent days of reverse learning, the dose was none Effect on the mean escape latencies. So put this Data suggests that FG 8094 assets the rats improved, a new spatial location of the hidden Platform to learn.

discussion

Benzodiazepine receptor agonists have anxiolytic and antispasmodic effects in humans and animals, but also induce amnesia. Conversely, inverse BG receptor agonists have anxiety-enhancing and anticonvulsant effects, but have cognitive-enhancing effects in living beings. BG receptor antagonists have no effect of their own, but block the effects of both BG receptor agonists and inverse BG receptor agonists. In contrast to full BZ receptor agonists, the present studies show that the selective inverse α ₅ -BZ receptor agonist FG 8094 has no anticonvulsant effects, but has cognitive enhancing effects in living beings.

The results of the in vivo binding experiments showed that FG 8094 at 1.0 mg / kg, virtually all α ₅ -containing receptors occupied less than 35% and 15% while the α _2/3 - or α ₁ receptors are occupied , Therefore, FG 8094 at 1.0 mg / kg is selective for α ₅ -containing receptors. At 10.0 mg / kg, FG 8094 significantly occupies receptors containing α ₁ , α ₂ and ₃ and consequently FG 8094 is not selective for receptors containing α ₅ at doses above 1.0 mg / kg.

In electrophysiological experiments it was demonstrated that FG 8094 is a full inverse receptor agonist against the α ₅ β ₂ γ ₂ receptor subtype, a weak partial inverse receptor agonist against receptor subtypes that contain α ₂ or α ₃ , and a Antagonist of α ₁ β ₂ γ ₂ , the main subunit combination in the brain. These data suggest that the main in vivo effects of FG 8094 are exerted on the α ₅ -containing subtype, which is predominantly located in the hippocampus.

In the MWM, rats are trained to be a submerged platform in a pool of cloudy Find water. The animals are believed to use the various visual cues attached to the walls of the room to guide their search strategy. Such a process requires flexible cognitive processing because the animal must first create a "map" of the space (ie, the spatial relationship between the stationary visual cues and the hidden platform) and then use the map to find its way to the hidden one To manage the platform. A well-trained rat that is brought into the pool at arbitrary locations swims more or less directly to the hidden platform, suggesting that it has detailed knowledge of the local area or a "cognitive map" and a new route to the hidden platform can plan with each new attempt.

In the present experiment, the consideration of 3a The mean latency (s) to find the hidden platform on consecutive training days suggests that the rats that received 0.3 and 1.0 mg / kg continuously passed the platform in less time than that Groups with vehicle or 0.1 mg / kg were found, although this effect did not reach statistical significance. During the probing attempt, there was no difference in the amount of time the control and drug groups spent looking for the quadrant in which the submerged platform had been placed during the training attempts, suggesting that there was no difference in the assets of the different groups to create and use a cognitive map. Similarly, there was no difference between the groups in their mean latency (s) to find a visible platform, indicating that FG 8094 had no effect on motor or sensory systems. However, on the first day of normal training, six days after the last attempt with a visible platform, the mean latency to find the hidden platform was significantly shorter for all groups who received FG 8094. These data imply that the spatial relationship between the position of the platform and the visual cues was better retained and / or recovered by the rats that received FG 8094 than by those who were given vehicles. In addition, when the hidden platform was relocated to a new location in the center of the Southwest Quadrant, rats that received 1.0 mg / kg FG 8094 were faster at learning this new location than rats that received vehicles.

In summary, FG 8094 is a selective inverse α ₅ -BZ receptor agonist that has no antispasmodic effects. In MWM, a hippocampus-dependent memory test, FG 8094 significantly increased the ability of rats to retain spatial information, suggesting that it improves cognitive processing.

Tabelle 1: Ki-Werte für Benzodiazepin-Stellen auf stabil transfizierten Zellen Table 1

Table 1: Ki values for benzodiazepine sites on stably transfected cells

The inhibition curves were generated with the aid of receptors which had been labeled with [ ³ H] Ro 15-1788 in a concentration twice the Kd value. The Ki values were calculated using the Cheng-Prussof equation. The data shown are mean values ± SEM for 3–6 determinations.

Tabelle 2: Inhibierung der [³H]Ro 15–1788-Bindung in vivo in drei Gehirnregionen nach FG 8094

Table 2: Inhibition of [ ³ H] Ro 15-1788 binding in vivo in three brain regions according to FG 8094

Mice were intraperitoneally FG 8094, 10 mg / kg or 1 mg / kg, 30 minutes before sacrifice and [ ³ H] Ro 15-1788 (0.1 mCi / g) injected. The brain regions were dissected, homogenized and filtered as described in the methods. The data are expressed as percent inhibition of binding relative to (vehicle-treated) control animals and are the mean ± SEM of 5 (10 mg / kg) or 10 (1 mg / kg) determinations.

Legend of the figures

1

Effects of flunitrazepam (1 μM), DMCM (1 μM) and FG 8094 (1 μM) on the GABA-EC ₂₀ current response in oocytes that expressed α ₁ β ₂ γ ₂ and α ₅ β ₂ γ ₂ . The data are expressed as the mean ± standard error of at least 4 separate oocytes.

2

Effects of FG 8094 (1 μM) on the GABA-EC ₂₀ current response in oocytes that express different α-subunit-containing receptors together with β ₂ γ ₂ . The data are expressed as the mean ± standard error of at least 4 separate oocytes.

3

The mean latency (s) by one submerged platform in the northeast quadrant of the Morris water maze to find on each training day (a), the time used up Search the northeast quadrant, expressed as a percentage of the total test length (60 s) (b), the mean latency (s), around a visible platform in the northeast quadrant of the water labyrinth on the last day of training with the visible one Find platform (c), the mean latency (s), to a submerged Platform in the northeast quadrant Reaching six days after the last training day (d) is for each treatment group after a 30 minute i.p. Pretreatment with vehicle or FG 8094 (doses as shown) shown * over a indicates that the Mean for this group differs significantly from the mean of the vehicle control group (post hoc Newman Keuls tests, p <0.05 assumed).

administration as pellets

A subsequent water maze experiment similar to that described above, except that a pellet of FG 8094 was implanted subcutaneously two hours before the start of the experiment and all training was done in one day, showed that FG 8094 again improved performance. In a second experiment, the vehicle or FG pellet rats were implanted exactly as described above. However, after the first four swimming attempts, the animals were injected (intravenously) with [ ³ H] FG 8094 and three minutes later the brains of the animals were quickly removed. In vivo binding showed that FG 8094 occupied about 85% of the α ₅ subtype-containing GABA _A receptors in the brain. A third experiment, identical to the second experiment, except that the animals were injected with [ ³ H] Ro 15-1788, showed that FG 8094 did not significantly occupy GABA _A receptors that did not contain the α ₅ subtype , This shows (i) that FG 8094 improved performance in the water maze test of spatial memory, (ii) that the dosing protocol used ensured that brain levels on FG 8094 were sufficient to contain 85% of the α ₅ -containing GABA _A - Occupy receptors without significantly occupying receptors that contained other subtypes. These data demonstrate that the inverse α ₅ receptor agonist is useful in neurological disorders with an associated cognitive defect and did not cause convulsions or sedation.

Claims (17)

Use of a compound which is an inverse GABA _A -α ₅ receptor agonist that is not a receptor agonist or inverse receptor agonist in GABA _A -α ₁ - and / or GABA _A -α ₂ - and / or GABA _A - α ₃ receptor is used to manufacture a medication to improve cognition in a person without causing cramps. Use according to claim 1, wherein the compound is not a receptor agonist or inverse receptor agonist for GABA _A -α ₁ and GABA _A -α ₂ and GABA _A -α ₃ receptors. Use according to claim 1 or 2, wherein the person a person with Alzheimer's disease is. Use according to claim 1, 2 or 3, wherein the cognitive enhancing compound is at least 25-fold selective for α ₅ -receptors against α ₁ -receptors. Use according to claim 1, 2, 3 or 4, wherein the cognitive-enhancing compound is at least 50-fold selective for α ₅ -containing receptors over α ₁ -, α ₂ - and α ₃ -containing receptors. Use according to any one of claims 1 to 5, wherein the cognitive enhancing compound has a Ki (nM) value of less than 2 against the α ₅ β ₃ γ ₂ receptor and more than 20 against the α ₁ β ₃ γ ₂ receptor , Use according to any one of claims 1 to 6, wherein the cognitive enhancing compound has a Ki (nM) value greater than 20 against the α ₂ β ₃ γ ₂ receptor. Use according to any one of the preceding claims, wherein the cognitive-enhancing link across the blood-brain barrier oral administration happens. A pharmaceutical composition comprising a compound and a pharmaceutically acceptable carrier therefor, the compound being an inverse GABA _A -α ₅ receptor agonist which is not a receptor agonist or inverse agonist for GABA _A -α ₁ - and / or GABA _A -α ₂ - and / or GABA _A -α ₃ receptors. A pharmaceutical composition according to claim 9, wherein the compound is not a receptor agonist or inverse receptor agonist at GABA _A -α ₁ and GABA _A -α ₂ and GABA _A -α ₃ receptors. Pharmaceutical composition according to claim 9 or 10, suitable for the treatment of Alzheimer's disease and the an effective amount of a compound with those in any of the Expectations 2 to 8 specified properties. A method of finding a compound capable of improving cognition without causing spasms, which method uses a GABA _A binding receptor to determine that the compound is an inverse α ₅ agonist and not an agonist or inverse agonist a, and / or α ₂ and / or α ₃ is included. The method of claim 12, wherein the compound is not a receptor agonist or inverse receptor agonist in GABA _A -α ₁ and GABA _A -α ₂ and GABA _A -α ₃ receptors. The method of claim 12 or 13, wherein the compound that in any of the claims 2 to 8 specified properties. A pharmaceutical composition comprising a A compound found using a method of claims 12, 13 or 14 and a pharmaceutically acceptable carrier therefor. Pharmaceutical composition according to claim 9, 10, 11 or 15, which is adapted for oral administration. A compound found according to claim 12, 13 or 14 was used for improving cognition.